Enhanced nanodrug delivery in tumors after near-infrared photoimmunotherapy

To date, the delivery of nanosized therapeutic agents to cancers largely relies on the enhanced permeability and retention (EPR) effects that are caused by the leaky nature of cancer vasculature. Whereas leaky vessels are often found in mouse xenografts, nanosized agents have demonstrated limited success in humans due to the relatively small magnitude of the EPR effect in naturally occurring cancers. To achieve the superior delivery of nanosized agents, alternate methods of increasing permeability and retention are needed. Near-infrared photoimmunotherapy (NIR-PIT) is a recently reported therapy that relies on an antibody-photon absorber conjugate that binds to tumors and then is activated by light. NIR-PIT causes an increase in nanodrug delivery by up to 24-fold compared to untreated tumors in which only the EPR effect is present. This effect, termed super-EPR (SUPR), can enhance the delivery of a wide variety of nanosized agents, including nanoparticles, antibodies, and protein-binding small-molecular-weight agents into tumors. Therefore, taking advantage of the SUPR effect after NIR-PIT may be a promising avenue to use a wide variety of nanodrugs in a highly effective manner

Disialoganglioside GD2-Targeted Near-Infrared Photoimmunotherapy (NIR-PIT) in Tumors of Neuroectodermal Origin

Disialoganglioside (GD2) is a subtype of glycolipids that is highly expressed in tumors of neuroectodermal origins, such as neuroblastoma and osteosarcoma. Its limited expression in normal tissues makes GD2 a potential target for precision therapy. Several anti-GD2 monoclonal antibodies are currently in clinical use and have had moderate success. Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that arms antibodies with IRDye700DX (IR700) and then exposes this antibody–dye conjugate (ADC) to NIR light at a wavelength of 690 nm. NIR light irradiation induces a profound photochemical response in IR700, resulting in protein aggregates that lead to cell membrane damage and death. In this study, we examined the feasibility of GD2-targeted NIR-PIT. Although GD2, like other glycolipids, is only located in the outer leaflet of the cell membrane, the aggregates formation exerted sufficient physical force to disrupt the cell membrane and kill target cells in vitro. In in vivo studies, tumor growth was significantly inhibited after GD2-targeted NIR-PIT, resulting in prolonged survival. Following GD2-targeted NIR-PIT, activation of host immunity was observed. In conclusion, GD2-targeted NIR-PIT was similarly effective to the conventional protein-targeted NIR-PIT. This study demonstrates that membrane glycolipid can be a new target of NIR-PIT

Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree

<div><p>Cholangiocarcinoma (CC) is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC) at the peribiliary gland (PBG) of large bile ducts and liver stem/progenitor cell (LPC) at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC) can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5) is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. <i>In vitro</i> experiments using siRNA for <i>SAMD5</i> revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines. <i>Conclusion</i>: SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.</p></div

Expression profiles of SAMD5 in human HCC and CC cell lines.

<p>(A) the relative expression of <i>SAMD5</i> gene in four CC cell lines (HuH28, TFK1, RBE and TKKK) and one HCC cell line (HuH7) to normal biliary epithelial cell (BEC) by quantitative RT-PCR. <i>SAMD5</i> mRNA was increased in all CC cell lines, but not expressed in HuH7. (n = 3; *<i>P</i> <0.05, compared to BEC) Data are mean ± standard error. n.d.: not detected (B). Immunocytochemical images of SAMD5 for CC cell lines. SAMD5 is visualized and localized at the nuclei of TKKK and RBE. Bars = 50 μm. (C and D) Images of exogenously introduced FLAG-tagged SAMD5 in HuH7 (C) and HuH28 (D) by Immunocytochemical staining. Overexpressed SAMD5 translocated to the nuclei of each cell. Bars = 50 μm.</p

Immunostaining of human liver tissue sections with anti-SAMD5 antibody.

<p>SAMD5 was stained for the paraffin-embedded sections of normal large bile duct at the hepatic hilum (A), intrahepatic CC (B), hilar CC (C and D), and extrahepatic CC (E). (A) Low or medial cytoplasmic staining of SAMD5 is observed in normal hilar large bile duct (asterisk). (Original magnification X200) (B) While the intrahepatic cholangiocytes show the cytoplasic staining of SAMD5 (arrow), the poorly-differentiated ICC exhibits striking nuclear staining of SAMD5. (Original magnification X100) (C) SAMD5 is stained in the nuclear of both poorly-differentiated ICC (p-ICC) and well-differentiated ICC (w-ICC) at the hepatic hilum. (Original magnification X200) (D) The cancerous cells invading hilar large bile duct show nuclear staining of SAMD5 (arrowhead). (Original magnification X200) (E) The papillary and moderately-differentiated tubular adenocarcinomas in the common bile duct exhibit nuclear staining of SAMD5. (Original magnification X200).</p

Primary antibodies.

<p>Primary antibodies.</p

Relationship between SAMD5 expression and cell cycle in CC cell line.

<p>(A) Real-time RT-PCR analysis of <i>SAMD5</i> mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of <i>SAMD5</i> in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of <i>SAMD5</i> mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *<i>P</i> <0.05; **<i>P</i> <0.01; ***<i>P</i> <0.001.</p

Result of cDNA microarray analysis of EpCAM⁺ cells between normal and DDC-fed mouse livers.

<p>Result of cDNA microarray analysis of EpCAM⁺ cells between normal and DDC-fed mouse livers.</p

IHC and PAS staining for serial sections of DDC-fed mice liver.

<p>(A) Immunostaining of SAMD5 and CK19 for DDC-fed mice liver. SAMD5 is markedly expressed in several PBGs (solid arrows) and columnar mucus-producing cholangiocytes (open arrows) at the hepatic hilum, whereas SAMD5 is not detected in cuboidal ductular cells (arrow heads). (B) PAS staining for the serial section of panel (A). Mucin is stained violet, while deposition of Iron and bile plug is observed as red and black agglutination. Mucin is detected in hilar large bile duct and PBG, but not in cuboidal ductular cells (arrow heads). The lower panel is a magnified image of the upper panel. Bars = 100 μm.</p